Several anticancer agents have biological effects which suggest they work by interfering with RNA synthesis and function. In this proposal, we would like to study some biological consequences of treatment with these agents. 5-fluorouracil (FUra) will be used as a model compound. FUra action can frequently be attributed to its conversion to fluorodeoxyuridylate (FdUMP) and inhibition of thymidylate synthetase, with subsequent inhibition of DNA biosynthesis. However, several laboratories have shown that incorporation of FUra into RNA can often be correlated with FUra's cytotoxic effect. Therefore, in some instances the effect of FUra on RNA synthesis and function may be related to its anti-cancer activity. FUra can inhibit ribosomal RNA (rRNA) maturation. Specific biochemical lesions of messenger RNA (mRNA) processing and function in mammalian systems have not been determined. We are studying this problem with a methotrexate (MTX)-resistant human cell line. The KB7D cell line is a clonally-derived, MTX-resistant cell line of human origin which overproduces dihydrofolate reductase (DHFR) and DHFR RNA. We have found during preliminary investigations that FUra exerts a large component of its cytotoxic effect upon the KB7D through its actions on RNA. In these cells, deoxythymidine (TdR) enhances the cytotoxic effect of FUra. Thus we are able to suppress the thymidylate synthetase site of FUra action by conducting studies in the presence of TdR. We have been able to develop an assay to monitor the effects of FUra on the levels of DHFR RNAs (i.e. mRNA and precursor RNAs). Initial experiments indicate that FUra increases the relative amount of DHFR RNA in KB7D cells during long-term exposure. This is the first demonstration that FUra can affect the levels of a particular mRNA species. We believe that this system will allow us to understand more fully the RNA-directed effect of FUra. Using this system, we should be able to determine the specific biochemical effects on mRNA maturation and function due to FUra. We should also be able to assess the effect of FUra incorporation into mRNA on the biochemical characteristics of the protein translation product, i.e. DHFR. These studies should serve as a model system to assess the value of numerous drugs which can affect RNA function and provide basic information about RNA metabolism.